: Expression of the cell death repressor, Bcl-2, has recently been shown to limit neuronal death in cerebral ischemia and is associated with ischemic tolerance. Current evidence suggests that Bcl-2 can function to either detoxify or decrease the production of reactive oxygen species in response to oxidative stress, or it may alter cellular Ca2+ transport processes. Bcl-2 is an integral membrane protein localized to sites of intracellular generation of free radicals including the mitochondrial membrane. Studies are proposed to address the mechanism of action of Bcl-2 in protection from hypoxia/reoxygenation injury, a model of cell death involving oxidative processes and Ca2+ overload. This study will test the hypotheses that: 1) in response to cellular hypoxia/reoxygenation, antioxidant activity of Bcl-2 can protect cells from delayed neural death; 2) mitochondrial respiratory inhibition resulting from hypoxia/reoxygenation injury precedes cell death and potentiates mitochondrial free radical production; 3) Bcl-2 potentiates the extent to which mitochondria can accumulate Ca2+ without respiratory impairment and without potentiation of reactive oxygen species generation. Methods used to test these hypotheses include the use of control and bcl-2 transfectants of GT1-7 cells (a hypothalamic neural cell line) exposed to chemical hypoxia/aglycemia, hypoxia achieved with oxygen replacement, pro-oxidants, or agents that dramatically increase cytosolic Ca2+. Measurements of rates of O2 consumption, free-radical and H2O2 production, pyridine nucleotide oxidation/reduction state, mitochondrial Ca2+ uptake by digitonin-permeabilized cells and mitochondria, cellular ATP, and determinations of lipid and protein oxidation will be performed. The study will contribute to current understanding of the mechanisms by which Bcl-2 rescues cells from death involving oxidative stress and pathological increases in Ca2+. The proposed work will further give insight into potential therapeutic approaches for the loss of neuronal function following ischemia/ reperfusion injury as well as other neurodegenerative disorders.